Populations of surface-associated bacteria are known as biofilms. The widespread recognition that biofilms impact myriad environments, from water pipes to indwelling devices in hospital patients, has led to an increased interest in investigating the molecular mechanisms underlying the formation and maintenance of these communities. A common feature emerges from all biofilms that have been analyzed to date: the constituent cells are held together by an extracellular matrix. Thus, regardless of the species under consideration, a central hypothesis emerges: Biofilm formation is a developmental process in which bacteria undergo a regulated lifestyle switch from a nomadic unicellular state to a sedentary multicellular state where different cell types co-exist and are spatially and temporally organized as a consequence of extracellular matrix production. The experiments proposed herein represent our continued efforts to critically test aspects of this central hypothesis using the sporulating bacterium Bacillus subtilis. Through a combination of genetic and biochemical approaches we have identified a key signal transduction pathway that governs the transition from single cells to matrix-enclosed communities where different cell types co-exist. Under biofilm-inducing conditions a subpopulation of cells begins to secrete the cyclic lipopeptide surfactin. Surfactin serves as a developmental signal that acts on a different subpopulation of cells - a process we refer to as paracrine signaling - inducing them to transcribe the genes involved in making the matrix. Through the production of the matrix the community develops a high degree of spatio-temporal organization culminating with sporulation occurring preferentially at the top of the biofilm. Entry into sporulation is tightly regulated - there is a developmental checkpoint whereby sporulation is delayed until the synthesis of the extracellular matrix is complete. Continuing to takes approaches that blend biochemical and molecular genetic methodologies we propose to: (1) characterize the protein component of the matrix, (2) define the mechanism responsible for the developmental checkpoint, and (3) determine the molecular mechanism of the surfactin-dependent signal transduction pathway. PUBLIC HEALTH RELEVANCE: Bacteria are remarkably adept at colonizing surfaces in a process known as biofilm formation. When biofilms form on the wrong surfaces, such as on open wounds or on implanted medical devices, they can cause chronic infections that are extremely difficult to erradicate. By studying the molecular details of how bacteria form biofilms we can devise strategies to interfere with the process; these could eventually be used to develop anti-biofilm agents to treat infections.